AMINO-ACID SUBSTITUTIONS IN THE MEMBRANE-BINDING DOMAIN OF CYTOCHROMEB(5) ALTER ITS MEMBRANE-BINDING PROPERTIES

The structure-function relationships of the 43-amino-acid membrane-bin ding domain of cytochrome b(5) have been examined in two mutant forms of the protein. In one mutant, two tryptophans in the membrane-binding domain, at positions 108 and 112, were replaced by leucines, and in t he second mutant, in addition, aspartic acid 103 was also replaced by leucine. The fluorescence emission spectra of the three proteins and t heir degree of quenching by brominated lipids indicate that the mutati ons are not producing major conformational changes or allowing a deepe r degree of penetration of the domain into the bilayer. The hydrophobi cities of the three proteins were compared, by determining strengths o f self-association and membrane affinities, and it was found that the protein with two additional leucines was much less hydrophobic and the one with three additional leucines was much more hydrophobic than the native cytochrome. It appears that small changes in amino acid compos ition, which produce no gross changes in the structure of the membrane -binding domain, will nevertheless produce very large changes in the s trengths of self- and membrane-association. These differences in self- association had profound effects on the times required for membrane-as sociation to reach equilibrium.